Cell models with inducible oncogenic translocations allow to evaluate the potential of drugs to favor secondary translocations
Authors: Anna Shmakova, Nikolai Lomov, Vladimir Viushkov, Tatyana Tsfasman, Yana Kozhevnikova, Darina Sokolova, Vadim Pokrovsky, Marina Syrkina, Diego Germini, Mikhail Rubtsov, Yegor Vassetzky
Journal: Cancer Communications (2022)

Dear Editor, Chromosomal translocations result from the interchange of genetic material between non-homologous chromosomes. Chromosomal translocations are formed by erroneous repair of double-stranded breaks (DSBs) via non-homologous end joining (NHEJ) [1]. Some genotoxic drugs produce DSBs and thus present a major risk factor for the development of oncogenic chromosomal translocations. The risk factors that interfere with translocation-prone DSB repair, once DSBs are already formed, are obscure, and potential effects of drugs on translocation formation during this step have never been explored. The study of chromosomal translocations is complicated since naturally occurring translocations are rare, and the localization of breakpoints varies from kilobases to hundreds of kilobases, which complicates their detection. In contrast, when DSBs are generated at precise loci, the translocation can be easily detected by PCR. In the present work, we developed two experimental human B cell-based models to study lymphomagenic t(8;14) MYC-IGH and leukemogenic t(8;21) AML1-ETO translocations, characteristic for Burkitt's lymphoma (BL) [2] and acute myeloid leukemia (AML), respectively [3]. We used these systems to study pathways and drugs that affect the probability of oncogenic translocations. BL often arises in people living with human immunodeficiency virus (HIV, PLWH) who are treated with combination antiretroviral therapy regimen [4], while chemotherapy is a risk factor for secondary AML [3]; therefore, we tested common antiretroviral and chemotherapeutic drugs for their ability to influence the rate of translocation formation in our systems. Two experimental systems for the targeted generation of DSBs in either the AML1 and ETO (iAML1-ETO cell line) or MYC and IGH loci (iMYC-IGH cell line) were created. The models were derived from the RPMI8866 lymphoblastoid cell line with the stable integration of the Cas9 gene, expressed under the control of a doxycycline (Dox)-inducible promoter and two guide RNA genes (targeting either AML1-ETO or MYC-IGH loci) (Figure 1A, Supplementary Figure S1). The DSB loci in the AML1-ETO model were similar to breakpoints in patients with therapy-related AML and cells treated with etoposide; the DSB loci in the MYC-IGH model were similar to breakpoints in patients with sporadic and HIV-induced BL (see Supplementary Methods for further information). The addition of Dox activated Cas9 expression after 4 hours (Figure 1B, Supplementary Figure S2) and stimulated the formation of DSBs in the selected loci (data not shown). The generated t(8;14) or t(8;21) translocations were detectable by qPCR using the primers that surrounded the translocation breakpoint (Figure 1C-D, Supplementary Figure S3). Translocations peaked after 48 hours for iMYC-IGH and 96 hours for iAML1-ETO (data not shown), and these timepoints were selected for further experiments. Without Dox treatment, chromosomal translocations were undetectable. To get insight into the mechanisms of the translocation generation in our system, we used several inhibitors of DSB repair pathways. iMYC-IGH cells were simultaneously treated with Dox and either Mirin (an MRE11 inhibitor), KU-55933 [(an ataxia telangiectasia mutated (ATM) inhibitor)], 17-N-allylamino-17-demethoxygeldanamycin [17-AAG, an Hsp90 inhibitor that inhibits homologous recombination (HR) repair by destabilizing Rad51], NU7026 [a DNA-dependent protein kinase (DNA-PK) inhibitor, classical NHEJ, (c-NHEJ)], or L67 [an inhibitor of DNA ligase I and III, alternative NHEJ (a-NHEJ)]. Treatment with Mirin, KU-55933 or L67 significantly decreased, whereas treatment with NU7026 significantly increased the MYC-IGH translocation rate (Figure 1E). Presumably, the inhibition of MRE11 and ATM, involved in the early steps of DNA damage detection and response, resulted in DSB repair defects [5] and ultimately cell cycle arrest or death, which decreased the overall translocation rates. ATM also promotes the clustering of DSBs into large repair foci [5], which might contribute to DSB proximity and translocations. The inhibition of c-NHEJ increased the translocation rate, while the inhibition of the a-NHEJ pathway decreased the translocation rate, which were in agreement with a previous studies [1]. The increase in the translocation rate when c-NHEJ is abrogated is due to the slow kinetics of DNA repair via a-NHEJ, which permits the free movement of unrepaired DNA ends and increases the chance of meeting their translocation partner in the nuclear space [1]. We next tested whether some chemotherapeutic drugs could influence the translocation formation and thus contribute to the formation of secondary neoplasms, e.g., therapy-related AML. We used drugs of different classes: platinum-based antineoplastics (carboplatin, cisplatin, oxaliplatin), topoisomerase inhibitors (doxorubicin, etoposide, irinotecan, mitoxantrone), antimetabolites (cytarabine, fluorouracil, methotrexate), poly(ADP-ribose) polymerase (PARP) inhibitor (olaparib), an alkylating agent (ifosfamide), and cytoskeletal drugs (docetaxel, paclitaxel). To exclude the effects related to cytotoxicity and cell death, we chose the non-lethal 10% inhibitory concentration (IC10) of the above drugs (Supplementary Table S3, Supplementary Figure S4). iAML1-ETO cells were simultaneously treated with Dox and chemotherapeutic drugs, and the level of t(8;21) translocations was measured and compared to those in Dox-treated cells. We found that the addition of methotrexate increased more than two-fold the rate of AML1-ETO translocations (Figure 1F). No significant differences were found for other drugs, which was further confirmed in iMYC-IGH cells with five other chemotherapeutic drugs used at IC10. No significant differences were found between the translocation rate in Dox-treated and Dox+drug-treated iMYC-IGH cells (Figure 1G). These results indicated that in the iMYC-IGH model, chemotherapeutic drugs did not interfere with the DSB repair once DSBs were formed. Thus, methotrexate use can increase the risk of secondary therapy-related AML, and patients treated with methotrexate should be monitored for the development of AML. Noteworthy, the development of therapy-related myelodysplastic syndrome/AML with t(8;21) and t(3;21) translocations was described in patients following low-dose treatment with methotrexate for rheumatoid arthritis [6]. We next tested whether antiretroviral drugs could influence the level of MYC-IGH translocations since BL is a common neoplasm in PLWH. We used drugs from different classes, such as nucleoside reverse transcriptase inhibitors (NRTIs) [abacavir (ABC), azidothymidine (AZT), emtricitabine (FTC), lamivudine (3TC)], non-NRTIs (nevirapine, rilpivirine), protease inhibitors (amprenavir, atazanavir, indinavir, saquinavir), and C-C motif chemokine receptor 5 (CCR5) antagonist (TAK779). The drugs were added simultaneously with Dox to the iMYC-IGH cell medium at the reported IC50 for virus inhibition. The chosen concentrations were not toxic for the cells (Supplementary Figure S4C). We found that two NRTIs, ABC and FTC, and CCR5 antagonist TAK779 significantly increased the rate of MYC-IGH translocations (Figure 1H). NTRIs are known to be incorporated into nuclear DNA by certain DNA polymerases during DNA repair, and they can act as chain terminators and directly inhibit cellular DNA polymerases by binding to their catalytic site [7]. Compromised DNA damage response may delay the classical repair machinery and engage translocation-prone a-NHEJ. Furthermore, mitochondrial toxicity of NRTIs could affect nuclear DNA integrity through reactive oxygen species production and imbalanced deoxynucleoside triphosphate pools [8]. CCR5 governs DNA damage repair (HR and single-strand annealing); consequently, CCR5 inhibitors were shown to sensitize cells to DNA-damaging agents [9]. Defects in HR and c-NHEJ repair mechanisms can result in chromosomal translocations [1]. A phase II clinical study of vicriviroc, a CCR5 antagonist, showed that vicriviroc treatment was associated with an increased risk of developing lymphomas [10]. Vicriviroc was not approved for HIV treatment. To conclude, we developed CRISPR/Cas9-based cell models with inducible AML1-ETO and MYC-IGH translocations to evaluate the potential of drugs to favor secondary translocations. In our screen, we identified four drugs, methotrexate, ABC, FTC and TAK779, that increased the rate of chromosomal translocations. A.S. and N.L. performed the following experiments: cell line creation and characterization, experimental setup, translocation rate analysis, survival analysis, RT-qPCR, western blotting, analyzed data and wrote the paper; D.S. and V.P. performed the following experiments: translocation rate and survival analysis and contributed new reagents; V.V., T.T., D.G. and Y.K. performed the following experiments: translocation rate analysis, RT-qPCR, western blotting; M.S. and M.R. analyzed data; Y.V. designed research, analyzed data and wrote the paper. This study was supported by Cancéropole IdF, the Koltzov Institute of Developmental Biology, Russian Academy of Sciences Government basic research programs (0088-2021-0007) and the Ministry of Science and Higher Education grants (075-15-2021-1075 to YV and 075-15-2021-1062 and 075-15-2021-1060 to MR); Russian Foundation for Basic Research (19-54-16002) and by the Interdisciplinary Scientific and Educational School of Moscow University «Molecular Technologies of the Living Systems and Synthetic Biology» to MR. AS was supported by the Travel Grant from Boehringer Ingelheim Fonds in 2018. The authors declare that they have no competing interests Methods and materials are available in the supplementary file. All data generated or analyzed during this study are included in this published article and its supplementary information files. 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